Summary of Work: This project applies biochemical and molecular biological approaches to the study of the complex interplay of the molecular signaling events that occur within T cells in response to engagement of the T cell antigen receptor (TCR) and co-stimulatory receptors. It is anticipated that a better understanding of the events that occur in the normal course of T cell activation will provide logical targets to probe with regard to their importance in immune pathogenicities, such as those characterized by immunosenescence in aged animals. The current focus is upon the kinases and phosphatases that regulate the phosphorylation status of key proteinaceous and phosphoinositide-based signaling molecules.Of particular interest has been the TCR-proximal protein tyrosine kinase (PTK) ZAP-70, which is required for T cell activation. By comparing the signaling pathways in Jurkat T cells that are either deficient or replete for ZAP-70 we have recently discovered that ZAP-70 regulates the activity of the PTK, Itk, which is involved in regulating intracellular concentrations of calcium ion, a key signaling molecule. The mechanism by which ZAP-70 regulates Itk activity is an indirect one, in that ZAP-70 phosphorylates the linker protein Lat, allowing Itk to form activating interactions with Lat and other proteins that associate with Lat as a consequence of Lat phosphorylation. These studies have helped to establish a new paradigm for signal transduction pathways in T lymphocytes.In the course of these studies two unexpected observations were made that have subsequently lead to important findings. The first observation is that signals can be sent through the TCR to downstream effector pathways in the absence of ZAP-70. This alternative signaling pathway is characterized by requiring extensive engagement of surface TCR and the production of weak/transient signals. Despite the weakness and transience of these signals, the existence of a TCR signaling pathway that can bypass ZAP-70 may be important in certain circumstances, perhaps, allowing a TCR signal to be transmitted in anergized T cells. The second unexpected observation made in these studies involves the constitutive mislocalization of Itk to the cell membrane in Jurkat T cells. Our attempts to understand why Itk was mislocalized in these cells lead to the observation that Jurkat T cells have a defect in the biochemical pathway that regulates the cellular concentration of the important signaling molecule phosphatidylinositol-3,4,5-P3 (PIP3). We have found that Jurkat T cells lack the tumor suppressor gene PTEN that usually regulates the levels of PIP3. In the absence of PTEN, PIP3 accumulates, even in unstimulated cells. This leads to the inappropriate recruitment of key signaling molecules, such as Itk to the cell membrane, priming them for activation in response to TCR stimulation. The importance of this observation stems from the predominant position that the Jurkat model system holds within the T cell signaling community, and how ignorance of this defect has lead to a gross underestimation of the important role that PIP3, and the enzymes that regulate its production, play in the process of TCR signaling.